Objectives: To analyse the changes in gene expression in a population of human periodontal ligament (HPDL) cells exposed to cyclic tensile strain in a three dimensional culture system. Results: Cells derived from extracted teeth were shown to represent a mixed population with greater than 95% of cells expressing markers characteristic of periodontal ligament fibroblasts with an oestobastic phenotype with a smaller sub-population (2-3%) of stem cells. Cells from several different donors were subjected to an applied tensile strain (5%) and compared to unstressed controls. RNA extracted immediately post-stress was analysed to determine changes in gene expression. Microarray data indicated a total of 164 genes responding to the applied strain, 38% of which have no known function. qRT-PCR analysis of HAS1, NR4A2, FOS, INHBA, FOSL2, RUNX2, ATF4, FOSL1, SP7, COL1A1, COL3A1, MMP1, MMP2, MMP3 and TIMP3 expression levels confirmed the microarray data. Further analysis of the data highlighted changes in expression of CLDN4, BCOR, PROK2 and INHBA genes known to be associated with inherited orofacial disorders. Conclusion: This study has shown that the periodontal ligament fibroblasts derived from extracted teeth and cultured in vitro in 3D 'in-vivo-like' constructs can be induced to respond to mechanical stimuli. The molecular events detected in periodontal ligament cells subjected to cyclic tensile strain direct the cell population to remodel the entire periodontium (cells and matrices) in a process involving cell migration. This study presents several new findings including the first report of the involvement of HAS1, NR4A2 and FOSL2 in the response of HPDL cells to mechanical stimulation, the first report for the of modulation CLDN4, BCOR, and PROK2 genes by mechanical perturbation, and the differential induction of SP7 isoforms by mechanical stimulation in HPDL.